Gas delivery configurations, foam control systems, and bag molding methods and articles for collapsible bag vessels and bioreactors

ABSTRACT

Systems for containing and manipulating fluids including systems and methods involving supported collapsible bags that may be used as reactors for performing chemical, biochemical and/or biological reactions contained therein are provided. Generally, a series of improvements and features for fluid containment systems such as gas delivery configurations, foam control systems and bag molding methods and articles for supported collapsible bag vessels and bioreactors are provided. For instance, in one aspect, fluids contained in a vessel can be sparged, e.g., such that a fluid is directed into a container of the vessel, and in some cases, the sparging can be controlled by rapidly activating or altering the degree of sparging as needed. Multiple spargers may be used in some cases. In another aspect, the vessel includes a seamless collapsible bag. In some cases, the collapsible bag may be injected, blown, or spin cast molded. In yet another aspect, the vessel includes a device which can reduce the foam produced or contained within the vessel. Sensors and/or controllers may optionally be used to monitor and/or control foaming.

FIELD OF INVENTION

The present invention relates generally to systems for containing andmanipulating fluids, and in certain embodiments, to systems and methodsinvolving collapsible bags that may be used as reactors for performingchemical, biochemical and/or biological reactions contained therein.

BACKGROUND

A variety of vessels for manipulating fluids and/or for carrying outchemical, biochemical and/or biological reactions are available. Forinstance, biological materials (e.g., animal and plant cells) including,for example, mammalian, plant or insect cells and microbial cultures canbe processed using bioreactors. Traditional bioreactors, which aretypically designed as stationary pressurized vessels, or disposablebioreactors, many of which utilize plastic sterile bags, may be used.Although reaction systems and other fluid manipulating systems (e.g.,mixing systems) are known, improvements to such systems would bebeneficial.

SUMMARY OF THE INVENTION

The present invention relates generally to systems for containing andmanipulating fluids, and in certain embodiments, to systems and methodsinvolving supported collapsible bags that may be used as bioreactors forperforming chemical, biochemical and/or biological reactions containedtherein. The subject matter of the present invention involves, in somecases, interrelated products, alternative solutions to a particularproblem, and/or a plurality of different uses of one or more systemsand/or articles.

In one aspect of the invention, a series of vessels are provided. In oneembodiment, a vessel configured to contain a volume of liquid isprovided. The vessel comprises a collapsible bag for containing thevolume of liquid, the collapsible bag having a volume of at least 2liters and a support structure surrounding and containing thecollapsible bag. The vessel also includes a first sparger connected tothe collapsible bag, the first sparger being in fluid communication witha source of a first gas composition, and a second sparger connected tothe collapsible bag, the second sparger being in fluid communicationwith a source of a second gas composition different from the first gascomposition.

In another embodiment, a vessel configured to contain a volume of liquidcomprises a collapsible bag able to contain the volume of liquid and asupport structure surrounding and containing the collapsible bag. Thevessel also includes a first sparger connected to the collapsible bag,the first sparger having a first aperture size, wherein at least aportion of the first sparger is dimensioned to be connected to a sourceof a first gas, and a second sparger connected to the collapsible bag,the second sparger having a second aperture size, wherein at least aportion of the second sparger is dimensioned to be connected to a sourceof a second gas.

In another embodiment, a vessel configured to contain a volume of liquidcomprises a container able to contain the volume of liquid. The vesselmay also include a magnetically-driven antifoaming device, at least aportion of which is positioned in a head space of the container when thecontainer contains the volume of liquid. The antifoaming device isconfigured and arranged to break up foam in the head space duringrotation of at least a portion of the antifoaming device.

In another embodiment, a vessel configured to contain a volume of liquidcomprises a collapsible bag able to contain the liquid and a reusablesupport structure surrounding and containing the collapsible bag. Thevessel also comprises a pressure sensor for determining a pressure inthe collapsible bag, the pressure sensor in fluid communication with thecollapsible bag, and an antifoaming device associated with thecollapsible bag and configured to break up foam in the collapsible bag.The vessel may also include a control system operatively associated withthe pressure sensor and the antifoaming device, wherein the controlsystem regulates the antifoaming device upon receipt of a signal fromthe pressure sensor.

In another embodiment, a collapsible bag configured to contain a volumeof at least 2 liters is provided. The collapsible bag comprises a firstrotatable impeller positioned at a bottom portion of the collapsiblebag, the first impeller able to be magnetically rotated, and a secondimpeller positioned at a top portion of the collapsible bag, the secondimpeller able to be magnetically rotated.

In another embodiment, a container able to contain a volume of liquid isprovided. The container comprises a collapsible bag having a volume ofat least 40 liters, wherein the collapsible bag does not include anyseams joining two or more flexible wall portions of the collapsible bag,and a reusable support structure surrounding and containing thecollapsible bag.

In another aspect of the invention, a series of methods are provided. Inone embodiment, a method comprises positioning a rigid component in amold having a shape configured to mold a container having a volume of atleast 10 mL, and introducing a first polymer or polymer precursor intothe mold. The method includes forming a seamless container within themold by solidifying the polymer or polymer precursor to form thecontainer, wherein the component is incorporated in the container.

In another embodiment, a method comprises introducing a first polymer orpolymer precursor into a mold, the mold having a shape configured tomold a container having a volume of at least 10 mL, the mold furthercomprising at least one mandrel for forming a functional component of aliquid containment system. The method also includes forming a containerwithin the mold, forming a component within the mold utilizing themandrel, and joining the functional component and the container withoutwelding.

In another embodiment, a method comprises introducing a first polymer orpolymer precursor into a mold, the mold having a shape configured tomold a collapsible bag having a volume of at least 10 mL and alsoconfigured to mold a base including a shaft configured to support amagnetic impeller and forming a collapsible bag within the mold. Themethod also includes introducing a second polymer precursor into themold, forming a component of the mixing system by solidifying the secondpolymer precursor, and joining the component of the mixing system andthe collapsible bag without welding.

In another embodiment, an article comprises a collapsible bag comprisinga flexible wall portion and a rigid portion comprising a base includinga shaft configured to support a magnetic impeller, wherein the rigidportion is embedded with the flexible wall portion.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 illustrates one embodiment of the invention, showing a containercontained within a support structure;

FIGS. 2A-2C illustrate techniques for forming a seamless container,according to another embodiment of the invention;

FIG. 3 illustrates a vessel for carrying out fluid manipulationsincluding biological, chemical, and biochemical processes, according toanother embodiment of the invention;

FIGS. 4A-4B illustrate various devices including impellers, according toanother embodiment of the invention;

FIG. 5 shows an impeller magnetically coupled to an external motor,according to another embodiment of the invention;

FIG. 6 shows an impeller, according to another embodiment of theinvention;

FIG. 7 shows an example of an antifoaming system, according to anotherembodiment of the invention;

FIG. 8 shows another example of an antifoaming system, according toanother embodiment of the invention; and

FIG. 9 shows an example of a control and feedback process, according toanother embodiment of the invention.

DETAILED DESCRIPTION

The present invention relates generally to systems for containing andmanipulating fluids, and in certain embodiments, to systems and methodsinvolving collapsible bags that may be used as reactors for performingchemical, biochemical and/or biological reactions contained therein.Generally, the invention provides a series of improvements and featuresfor fluid containment systems such as gas delivery configurations, foamcontrol systems and bag molding methods and articles for supportedcollapsible bag vessels and bioreactors. For instance, in one aspect,fluids contained in a vessel can be sparged, e.g., such that a fluid isdirected into a container of the vessel, and in some cases, the spargingcan be controlled by rapidly activating or altering the degree ofsparging as needed. Multiple spargers may be used in some cases. Inanother aspect, the vessel includes a seamless collapsible bag. In somecases, the collapsible bag may be injected, blown, or spin cast molded.In yet another aspect, the vessel includes a device which can reduce thefoam produced or contained within the vessel. Sensors and/or controllersmay optionally be used to monitor and/or control foaming.

The following documents are incorporated herein by reference: U.S.Provisional Patent Application Ser. No. 60/903,977, filed Feb. 28, 2007,entitled “Weight Measurements of Liquids in Flexible Containers,” by P.A. Mitchell, et al.; U.S. patent application Ser. No. 11/147,124, filedJun. 6, 2005, entitled “Disposable Bioreactor Systems and Methods,” byG. Hodge, et al., published as U.S. Patent Application Publication No.2005/0272146 on Dec. 8, 2005; International Patent Application No.PCT/US2005/020083, filed Jun. 6, 2005, entitled “Disposable BioreactorSystems and Methods,” by G. Hodge, et al., published as WO 2005/118771on Dec. 15, 2005; and International Patent Application No.PCT/US2005/002985, filed Feb. 3, 2005, entitled “System and Method forManufacturing,” by G. Hodge, et al., published as WO 2005/076093 on Aug.18, 2005.

Although much of the description herein involves an exemplaryapplication of the present invention related to bioreactors (and/orbiochemical and chemical reaction systems), the invention and its usesare not so limited, and it should be understood that aspects of theinvention can also be used in other settings, including those involvingcontainment systems in general, as well as systems for containmentand/or processing of a fluid in a container (e.g., mixing systems). Itshould also be understood that while many examples provided hereininvolve the use of collapsible bags or flexible containers, aspects ofthe invention can be integrated with systems involving non-collapsiblebags, rigid containers, and other configurations involving liquidcontainment.

In one aspect, vessels configured to contain a volume of liquid areprovided. In certain embodiments, the vessels are a part of a bioreactorsystem. For example, a non-limiting example of a bioreactor systemincluding a container, such as a flexible container, is shown in theschematic diagram of FIG. 1. As shown in the embodiment illustrated inFIG. 1, vessel 10 includes a reusable support structure 14 (e.g., astainless steel tank) that surrounds and contains a container 18. Insome embodiments, the container is configured as a collapsible bag(e.g., a polymeric bag). Additionally and/or alternatively, all orportions of the collapsible bag or other container may comprise asubstantially rigid material such as a rigid polymer, metal, and/orglass. In other embodiments, a rigid container is used in thisconfiguration. The container may be disposable and may be configured tobe easily removable from the support structure. In some embodiments, thecontainer is non-integrally connected to the support structure. As usedherein, the term “integrally connected,” when referring to two or moreobjects, means separation of the two or more objects requires causingdamage to at least one of the object (or components of the object), forexample, by breaking or peeling (e.g., separating components fastenedtogether via adhesives, tools, etc.).

If a collapsible bag is used, the collapsible bag may be constructed andarranged for containing a liquid 22, which may contain reactants, media,and/or other components necessary for carrying out a desired processsuch as a chemical, biochemical and/or biological reaction. Thecollapsible bag may also be configured such that liquid 22 remainssubstantially in contact only with the collapsible bag during use andnot in contact with support structure 14. In such embodiments, the bagmay be disposable and used for a single reaction or a single series ofreactions, after which the bag is discarded. Because the liquid in thecollapsible bag may not come into contact with the support structure,the support structure can be reused without cleaning. That is, after areaction takes place in container 18, the container can be removed fromthe support structure and replaced by a second (e.g., disposable)container. A second reaction can be carried out in the second containerwithout having to clean either the first container or the reusablesupport structure.

Also shown in FIG. 1 are an optional inlet port 42 and optional outletport 46, which can be formed in the container and/or reusable supportstructure and can facilitate more convenient introduction and removal ofa liquid and/or gas from the container. The container may have anysuitable number of inlet ports and any suitable number of outlet ports.For example, a plurality of inlet ports may be used to provide differentgas compositions (e.g., via a plurality of spargers 47), and/or to allowseparation of gases prior to their introduction into the container.These ports may be positioned in any suitable location with respect tocontainer 18. For instance, for certain vessels including spargers, thecontainer may include one more gas inlet ports located at a bottomportion of the container. Tubing may be connected to the inlet and/oroutlet ports to form, e.g., delivery and harvest lines, respectively,for introducing and removing liquid from the container. Optionally, thecontainer and/or support structure may include a utility tower 50, whichmay be provided to facilitate interconnection of one or more devicesinternal to the container and/or support structure with one or morepumps, controllers, and/or electronics (e.g., sensor electronics,electronic interfaces, and pressurized gas controllers) or otherdevices. Such devices may be controlled using a control system 34.

For systems including multiple spargers, control system 34 may beoperatively associated with each of the spargers and configured tooperate the spargers independently of each other. This can allow, forexample, control of multiple gases being introduced into the container.

The vessel may optionally include a mixing system such as an impeller51, which can be rotated (e.g., about a single axis) using a motor 52that can be external to the container. In some embodiments, as describedin more detail below, the impeller and motor are magnetically coupled.The mixing system can be controlled by control system 34. Mixing systemsare described in further detail below.

Additionally and/or alternatively, the vessel may include an antifoamingsystem such as a mechanical antifoaming device. As shown in theembodiment illustrated in FIG. 1, an antifoaming device may include, forexample, an impeller 61 that can be rotated (e.g., magnetically) using amotor 62, which may be external to the container. The impeller can beused to collapse a foam contained in a head space 63 of the container.In some embodiments, the antifoaming system is in electricalcommunication with a sensor 43 (e.g., a foam sensor) via control system34. The sensor may determine, for instance, the level or amount of foamin the head space or the pressure in the container, which can triggerregulation or control of the antifoaming system. In other embodiments,the antifoaming system is operated independently of any sensors.

The support structure and/or the container may also include, in someembodiments, one or more ports 54 that can be used for sampling,analyzing (e.g., determining pH and/or amount of dissolved gases in theliquid), or for other purposes. The support structure may also includeone or more site windows 60 for viewing a level of liquid within thecontainer. One or more connections 64 may be positioned at a top portionof the container or at any other suitable location. Connections 64 mayinclude openings, tubes, and/or valves for adding or withdrawingliquids, gases, and the like from the container, each of which mayoptionally include a flow sensor and/or filter (not shown). The supportstructure may further include a plurality of legs 66, optionally withwheels 68 for facilitating transport of the vessel.

It should be understood that not all of the features shown in FIG. 1need be present in all embodiments of the invention and that theillustrated elements may be otherwise positioned or configured. Also,additional elements may be present in other embodiments, such as theelements described herein.

Various aspects of the present invention are directed to a vesselincluding a container such as a collapsible bag. “Flexible container”,“flexible bag”, or “collapsible bag” as used herein, indicates that thecontainer or bag is unable to maintain its shape and/or structuralintegrity when subjected to the internal pressures (e.g., due to theweight and/or hydrostatic pressure of liquids and/or gases containedtherein expected during operation) without the benefit of a separatesupport structure. The collapsible bag may be made out of inherentlyflexible materials, such as many plastics, or may be made out of whatare normally considered rigid materials (e.g., glass or certain metals)but having a thickness and/or physical properties rendering thecontainer as a whole unable to maintain its shape and/or structuralintegrity when subjected to the internal pressures expected duringoperation without the benefit of a separate support structure. In someembodiments, collapsible bags include a combination of flexible andrigid materials; for example, the bag may include rigid components suchas connections, ports, supports for a mixing and/or antifoaming system,etc.

The container (e.g., collapsible bag) may have any suitable size forcontaining a liquid. For example, the container may have a volumebetween 1-40 L, 40-100 L, 100-200 L, 200-300 L, 300-500 L, 500-750 L,750-1,000 L, 1,000-2,000 L, 2,000-5,000 L, or 5,000-10,000 L. Volumesgreater than 10,000 L are also possible.

In some embodiments, the collapsible bag is disposable and is formed ofa suitable flexible material. The flexible material may be one that isUSP Class VI certified, e.g., silicone, polycarbonate, polyethylene, andpolypropylene. Non-limiting examples of flexible materials includepolymers such as polyethylene (e.g., linear low density polyethylene andultra low density polyethylene), polypropylene, polyvinylchloride,polyvinyldichloride, polyvinylidene chloride, ethylene vinyl acetate,polycarbonate, polymethacrylate, polyvinyl alcohol, nylon, siliconerubber, other synthetic rubbers and/or plastics. As noted above,portions of the flexible container may comprise a substantially rigidmaterial such as a rigid polymer (e.g., high density polyethylene),metal, and/or glass (e.g., in areas for supporting fittings, etc.). Inother embodiments, the container is substantially rigid material. All orportions of the container may be optically transparent to allow viewingof contents inside the container. The materials or combination ofmaterials used to form the container may be chosen based on one or moreproperties such as flexibility, puncture strength, tensile strength,liquid and gas permeabilities, opacity, and adaptability to certainprocesses such as blow molding, injection molding, or spin cast molding(e.g., for forming seamless collapsible bags).

The container (e.g., collapsible bag) may have any suitable thicknessfor holding a liquid and may be designed to have a certain resistance topuncturing during operation or while being handled. For instance, thewalls of a container may have a total thickness of less than or equal to250 mils (1 mil is 25.4 micrometers), less than or equal to 200 mils,less than or equal to 100 mils, less than or equal to 70 mils (1 mil is25.4 micrometers), less than or equal to 50 mils, less than or equal to25 mils, less than or equal to 15 mils, or less than or equal to 10mils. In some embodiments, the container includes more than one layer ofmaterial that may be laminated together or otherwise attached to oneanother to impart certain properties to the container. For instance, onelayer may be formed of a material that is substantially oxygenimpermeable. Another layer may be formed of a material to impartstrength to the container. Yet another layer may be included to impartchemical resistance to fluid that may be contained in the container. Itshould be understood that a container may be formed of any suitablecombinations of layers and that the invention is not limited in thisrespect. The container (e.g., collapsible bag) may include, for example,1 layer, greater than or equal to 2 layers, greater than or equal to 3layers, or greater than equal to 5 layers of material(s). Each layer mayhave a thickness of, for example, less than or equal to 200 mils, lessthan or equal to 100 mils, less than or equal to 50 mils, less than orequal to 25 mils, less than or equal to 15 mils, less than or equal to10 mils, less than or equal to 5 mils, or less than or equal to 3 mils,or combinations thereof.

In one set of embodiments of the invention, the container is seamless.The container may be, for example, a seamless collapsible bag or aseamless rigid (or semi-rigid) container. Many existing collapsible bagsare constructed from two sheets of a plastic material joined by thermalor chemical bonding to form a container having two longitudinal seams.The open ends of the sheets are then sealed using known techniques andaccess apertures are formed through the container wall. During use,collapsible bags having seams can cause the formation of crevices at ornear the seams where fluids or reagents contained therein are notthoroughly mixed. In certain embodiments involving, for example, the useof collapsible bags for performing a chemical, biochemical and/orbiological reaction, unmixed reagents can cause a reduction in yield ofa desired product. The presence of the seams in a collapsible bag canalso result in the inability of the collapsible bag to conform to theshape of a reusable support structure that may support the bag. By usingcollapsible bags without any seams joining two or more flexible wallportions of the bag, however, the problems of mixing and conformity maybe avoided or reduced. In certain embodiments, seamless collapsible bagscan be made specifically to fit a particular reusable support structurehaving a unique shape and configuration. Substantially perfect-fittingcollapsible bags can be used, for example, as part of a bioreactorsystem or a biochemical and/or chemical reaction system. Seamless rigidor semi-rigid containers may also be beneficial in some instances.

In certain embodiments, a collapsible bag that does not include anyseams joining two or more flexible wall portions of the collapsible bag(i.e., a seamless collapsible bag) has a certain volume for containing aliquid. The seamless collapsible bag may have a volume of, for example,at least 1 L, at least 10 liters, at least 20 liters, at least 40liters, at least 50 liters, at least 70 liters, at least 100 liters, atleast 150 liters, at least 200 liters, at least 300 liters, at least 500liters, at least 700 liters, or at least 1,000 liters. Seamlesscollapsible bags may also have volumes greater than 1,000 liters (e.g.,1,000-5,000 liters or 5,000-10,000 liters) as needed. In someembodiments, the collapsible bag is positioned in a reusable supportstructure for surrounding and containing the flexible container.

In one embodiment, a seamless collapsible bag is formed in a process inwhich the bag liner (e.g., the flexible wall portions of the bag), aswell as one or more components such as a component of an agitator/mixersystem (e.g., a shaft and/or a support base), port, etc. is cast fromone continuous supply of a polymeric precursor material. In some cases,the casting may occur without hermetically sealing, e.g., via welding.Such a seamless collapsible bag may allow the interior liquid or otherproduct to contact a generally even surface, e.g., one which does notcontain substantial wrinkles, folds, crevices, or the like. In addition,in some cases, the collapsible bag complementarily fits within a supportstructure when installed and filled with a liquid or product. Theseamless collapsible bag may also have a generally uniform polymericsurface chemistry which may, for example, minimize side reactions.Methods of forming seamless collapsible bags involving more than onepolymeric precursor materials can also be performed.

Seamless collapsible bags can be created by a variety of methods. In oneembodiment, a seamless collapsible bag is formed by injecting liquidplastic into a mold that has been pre-fitted with components such asports, connections, supports, and rigid portions configured to support amixing system (e.g., a shaft and/or a base) that are subsequentlysurrounded, submerged, and/or embedded by the liquid plastic. Thecomponent may be a rigid component, e.g., one that can substantiallymaintain its shape and/or structural integrity during use. Any suitablenumber of components (e.g., at least 1, 2, 5, 10, 15, etc.) can beintegrated with containers (e.g., collapsible bags) using methodsdescribed herein. The mold may be designed to form a collapsible baghaving the shape and volume of the mold, which may have a substantiallysimilar shape, volume, and/or configuration of a reusable supportstructure.

In one embodiment, the container is formed by using an embeddedcomponent/linear molding (ECM) technique. In one such technique, rigidor pre-made components such as tube ports, agitator bases, etc. arefirst positioned in the mold. A polymer or polymer precursor used toform a container (e.g., a seamless collapsible bag) may be introduced(e.g., in a melt state) via a polymer fabrication technique such asthose described below. In some cases, a component or a portion of thecomponent is partially melted by the polymer precursor, allowing thecomponent to form a continuous element with the container. That is, thecomponent can be joined (e.g., fused) with one or more wall portions ofthe container (e.g., flexible wall portions of a collapsible bag) toform a single, integral piece of material without seams.

Referring now to FIG. 2A, an illustrative example of this process isshown. In this figure, a premounted component 202 is held between moldwalls 205 and 206. Polymer precursor 210 is then introduced into themold via any suitable technique. The polymer precursor then flows aroundcomponent 202 and is hardened, solidified, set, or cured, therebyforming a seamless connection between the polymer itself and theembedded component.

In some cases, components are designed with thinner portions that can bemelted with a polymer precursor (e.g., in the melt state) duringformation of a container. For example, as shown in the embodimentillustrated in FIG. 2B, component 203, a rigid portion including arecess 212 into which at least a portion of a drive head (not shown) ofa mixing and/or antifoaming system can be inserted, is positioned in amold comprising a shape configured to mold a container. Polymerprecursor 210 is then introduced into the mold via any suitabletechnique. The polymer precursor then flows around component 203 andmelts thinner portions 204, causing fusion of portions of the walls ofthe container and portions of the component. The container and componentare then hardened, solidified, set, or cured, thereby forming a single,integral piece of material where a seamless connection exists betweenthe polymer itself and the embedded component. This technique can beused to form, for example, a container (e.g., a seamless collapsiblebag) having a volume of greater than, e.g., 40 L, 50 L, 100 L, 200 L,1,000 L, etc., and having embedded therein components such as one ormore support bases and/or shafts for impellers of a mixing system(s).

Accordingly, one embodiment of the invention includes the method ofjoining together a wall portion of a container and at least a portion ofa functional component during formation of the container within a mold,wherein the portion of the functional component is melted during thejoining step. The wall portion of the container may have a firstthickness and the portion of the functional component may have a secondthickness, the thicknesses being within, for example, less than 100%,80%, 60%, 40%, 20%, 10%, or 5% of each other, relative to the larger ofthe first and second thicknesses.

In another embodiment, the container may be formed using a continuouscomponent/liner molding (CCM) technique. In one such technique, acollapsible bag or other container is cast de novo from a polymer orpolymer precursor stream. The polymer or polymer precursor used to formthe seamless collapsible bag is introduced via a polymer fabricationtechnique such as those described below. Components can be introducedinto the flexible container by using mandrels, barriers, baffles, andthe like to direct the polymer precursor to form functional componentsof a liquid containment system such as tube ports and agitator bases as,for example, one continuous polymer. After setting or curing of thepolymer or polymer precursor, the mandrels, barriers, etc., may bewithdrawn. For example, as is shown in FIG. 2C, mandrel 209 is heldbetween mold walls 205 and 206. Polymer 210 is then introduced into themold via any suitable technique, such as those described below. Thepolymer then flows around mandrel 209 and is hardened or set around themandrel. The mandrel can then be subsequently withdrawn. This techniquecan be used to form, for example, a container (e.g., a seamlesscollapsible bag) having a volume of greater than, e.g., 40 L, 50 L, 100L, 200 L, 1,000 L, etc., having combined therein components (e.g., rigidcomponents) that are joined to the collapsible bag without welding.

Combinations of these and/or other techniques may also be used in otherembodiments. For instance, in some cases, different polymer formulations(such as low molecular weight polyethylene, high molecular weightpolyethylene, polypropylene, silicone, polycarbonate, polymethacrylate,combinations thereof or precursors thereof) can be simultaneouslyinjected into regions of the mold designed to form a more rigidstructure such as tubing or sensor ports, agitation systems, etc. In oneparticular embodiment, a method involves introducing a first polymer orpolymer precursor into a mold comprising a shape configured to mold acollapsible bag having a volume of at least 10 mL, 1 L, 40 L, 100 L, or1,000 L, etc. The mold may further comprise a shape configured to mold acomponent of a mixing and/or antifoaming system such as a shaft and/orbase configured to support an impeller. The method may also includeintroducing a second polymer or polymer precursor into the mold to moldthe component of the mixing system. Accordingly, the component of themixing system and the collapsible bag may be joined without weldingusing methods described herein. In some instances, the first and secondpolymers or polymer precursors are introduced simultaneously. The firstand second polymers or polymer precursors may be the same in someembodiments, or different in other embodiments. Such a method can beused to form, for example, base plates for mixing/agitation systems,antifoaming systems, or other components. In other embodiments, a numberof polymers can be introduced into the mold (e.g., simultaneously) toform containers with multiple components.

As mentioned, a polymer or polymer precursor may be introduced into amold to form a container such as a collapsible bag (e.g., a seamlesscollapsible bag) using any suitable technique. For instance, in oneembodiment, the collapsible bag may be fabricated via a spin castingprocess. For example, during spin casting, a mold may be spun duringinjection of the polymer or polymeric precursor to deposit a uniformcoating of plastic on the mold surface. In another embodiment, thecollapsible bag is fabricated via an injection molding process. Forinstance, the polymeric precursor may be pumped into the space betweenan inner mold and the outer mold. In yet another embodiment, thecollapsible bag can be fabricated via a blow molding process. Thepolymer may be deposited, for example, via a gas injection, to expandthe polymer against the mold surface. In yet another embodiment, acombination of these and/or other techniques may be used. Those ofordinary skill in the art will be familiar with polymer processingtechniques such as spin casting, injection molding, and/or blow molding,and will be able to use such techniques in the methods of the presentinvention as described herein to prepare suitable collapsible bags orother containers.

Although many embodiments herein describe seamless collapsible bags, insome embodiments, collapsible bags or other containers described hereincan be fabricated with seams between flexible wall portions of thecontainer. In other embodiments, collapsible bags or other containerscan be fabricated with seams between a component and one or moreflexible wall portions of the container. The act of joining two or morewall portions or a wall portion and a portion of a component may beachieved by methods such as welding (e.g., heat welding and ultrasonicwelding), bolting, use of adhesives, fastening, or other attachingtechniques. Combination of seams and seamless connections can also befabricated. It should also be understood that while many of the methodsdescribed herein refer to fabrication of collapsible bags, the methodsmay also be applied to rigid containers. The methods described hereinused to form containers such as collapsible bags (e.g., bags with orwithout seams) may be adapted to include components of various sizes.For instance, although the flexible wall portions of a collapsible bagmay having a thickness of, for example, less than or equal to 100 mils,less than or equal to 70 mils, less than or equal to 50 mils, less thanor equal to 25 mils, less than or equal to 15 mils, or less than orequal to 10 mils, a component to be incorporated with the container mayhave a thickness or a height of, for example, greater than 0.5 mm,greater than 1 cm, greater than 1.5 cm, greater than 2 cm, greater than5 cm, or greater than 10 cm. In some cases, the component has at leastone cross-sectional dimension (e.g., a height, length, width, ordiameter) of, for example, greater than 0.5 mm, greater than 1 cm,greater than 1.5 cm, greater than 2 cm, greater than 5 cm, greater than10 cm, greater than 15 cm, greater than 20 cm, greater than 25 cm, orgreater than 30 cm. In certain embodiments, the thickness of acollapsible bag (or other container) and the thickness of a portion of acomponent to be joined (e.g., fused) with the collapsible bag are within30%, 20%, 15%, 10% or 5% of each other (relative to the thickestportion). This matching of thicknesses can aid joining (e.g., melting,welding, etc.) of the materials, as described in more detail below.

Components that are integrated with collapsible bags or other containersmay be formed in any suitable material, which may be the same or adifferent material from that of the bag or container. For instance, inone embodiment, a container is formed in a first polymer and a componentis formed in a second polymer that is different (e.g., having adifferent composition, molecular weight, and/or chemical structure,etc.) from the first polymer. Those of ordinary skill in the art will befamiliar with material processing techniques and will be able to usesuch techniques in the methods described herein to choose suitablematerials and combinations of materials.

In some embodiments, components that are integrated with collapsiblebags or other containers using methods described herein are formed inone or more materials that is/are USP Class VI certified, e.g.,silicone, polycarbonate, polyethylene, and polypropylene. Non-limitingexamples of materials that can be used to form components includepolymers such as polyethylene (e.g., low density polyethylene and highdensity polyethylene), polypropylene, polyvinylchloride,polyvinyldichloride, polyvinylidene chloride, ethylene vinyl acetate,polyvinyl alcohol, polycarbonate, polymethacrylate, nylon, siliconerubber, other synthetic rubbers and/or plastics, and combinationsthereof. Ceramics, metals, and magnetic materials can also be used toform all or portions of a component. In some embodiments, all orportions of a component are rigid; in other embodiments, all or portionsof a component are flexible. The material(s) used to form a componentmay be chosen based on, for example, the function of the componentand/or one or more properties such as compatibility with the container,flexibility, tensile strength, hardness, liquid and gas permeabilities,and adaptability to certain processes such as blow molding, injectionmolding, or spin cast molding.

In certain embodiments, especially in certain embodiments involvingfluid manipulations or carrying out a chemical, biochemical and/orbiological reaction in a container (e.g., a collapsible bag), thecontainer is substantially closed, e.g., the container is substantiallysealed from the environment outside of the container except, in certainembodiments, for one or more inlet and/or outlet ports that allowaddition to, and/or withdrawal of contents from, the container. If acollapsible bag is used, it may be substantially deflated prior to beingfilled with a liquid, and may begin to inflate as it is filled withliquid. In other embodiments, aspects of the invention can be applied toopened container systems.

In some cases, fluids may be introduced and/or removed from a vessel viainlet ports and/or outlet ports. The vessel may be a part of a reactorsystem for performing a biological, biochemical, or chemical reaction.As mentioned, a container (e.g., a collapsible bag), which may be partof the vessel, may have any suitable number of inlet ports and anysuitable number of outlet ports. In some cases, pumps, such asdisposable pumps, may be used to introduce a gas or other fluid into thecontainer, e.g., via an inlet port, and/or which may be used to remove agas or other fluid from the container, e.g., via an outlet port. Forinstance, a magnetically-coupled pump may be created by encasing adisposable magnetic impeller head in an enclosure with inlet(s) andoutlet(s) that achieves fluid pumping. Flexible blades may be used toenhance pumping or provide pressure relief. In another embodiment,pumping of fluids, gas and/or powder may be achieved without pump headsand/or pump chambers by sequentially squeezing, for example, anelectromechanical-polymeric tube that effectively achieves“peristalsis.” One way valves in the tube may optionally be used, whichmay aid in the prevention of backflow.

Certain aspects of the present invention also include a supportstructure, for example, support structure 14 as shown in FIG. 1, whichcan surround and contain container 18. The support structure may haveany suitable shape able to surround and/or contain the container. Insome cases, the support structure is reusable. The support structure maybe formed of a substantially rigid material. Non-limiting examples ofmaterials that can be used to form the reusable support structureinclude stainless steel, aluminum, glass, resin-impregnated fiberglassor carbon fiber, polymers (e.g., high-density polyethylene,polyacrylate, polycarbonate, polystyrene, nylon or other polyamides,polyesters, phenolic polymers, and combinations thereof. The materialsmay be certified for use in the environment in which it is used. Forexample, non-shedding materials may be used in environments whereminimal particulate generation is required.

In some embodiments, the reusable support structure may be designed tohave a height and diameter similar to standard stainless steelbioreactors (or other standard reactors or vessels). The design may alsobe scaleable down to small volume bench reactor systems. Accordingly,the reusable support structure may have any suitable volume for carryingout a desired chemical, biochemical and/or biological reaction. In manyinstances, the reusable support structure has a volume substantiallysimilar to that of the container. For instance, a single reusablesupport structure may be used to support and contain and singlecontainer having a substantially similar volume. In other cases,however, a reusable support structure is used to contain more than onecontainer. The reusable support structure may have a volume between, forexample, 1-100 L, 100-200 L, 200-300 L, 300-500 L, 500-750 L, 750-1,000L, 1,000-2,000 L, 2,000-5,000 L, or 5,000-10,000 L. Volumes greater than10,000 L are also possible.

In other embodiments, however a vessel of the present invention does notinclude a separate container (e.g., collapsible bag) and supportstructure, but instead comprises a self-supporting disposable container.The container may be, for example, a plastic vessel and, in some cases,may include an agitation system integrally or removably attachedthereto. The agitation system may be disposable along with thecontainer. In one particular embodiment, such a system includes animpeller welded or bolted to a polymeric container. It should thereforebe understood that many of the aspects and features of the vesselsdescribed herein with reference to a container and a support structure(for example, a seamless container, a sparging system, an antifoamingdevice, etc.), are also applicable to a self-supporting disposablecontainer.

As an example, a container, such as container 18 shown in FIG. 3, mayinclude various sensors and/or probes for controlling and/or monitoringone or more process parameters inside the disposable container such as,for example, temperature, pressure, pH, dissolved oxygen (DO), dissolvedcarbon dioxide (DCO₂), mixing rate, and gas flow rate. The sensor mayalso be an optical sensor in some cases.

In some embodiments, process control may be achieved in ways which donot compromise the sterile barrier established by a disposablecontainer. For example, gas flow may be monitored and/or controlled by arotameter or a mass flow meter upstream of an inlet air filter. Inanother embodiment, disposable optical probes may be designed to use“patches” of material containing an indicator dye which can be mountedon the inner surface of the disposable container and read through thewall of the disposable container via a window in the reusable supportstructure. For example, dissolved oxygen, pH, and/or CO₂ each may bemonitored and controlled by an optical patch and sensor mounted on,e.g., a gamma-irradiatable, biocompatible polymer which, can be sealedto, embedded in, or otherwise attached to the surface of the container.

As a specific example of a sensor, as shown in the embodimentillustrated in FIG. 3, container 18 may be operatively associated with atemperature controller 106 which may be, for example, a heat exchanger,a closed loop water jacket, an electric heating blanket, or a Peltierheater. Other heaters for heating a liquid inside a container are knownto those of ordinary skill in the art and can also be used incombination with container 18. The heater may also include athermocouple and/or a resistance temperature detector (RTD) for sensinga temperature of the contents inside the container. The thermocouple maybe operatively connected to the temperature controller to controltemperature of the contents in the container. Optionally, aheat-conducting material may be embedded in the surface of the containerto provide a heat transfer surface to overcome the insulating effect ofthe material used to form other portions of the container.

In general, as used herein, a component of an inventive system that is“operatively associated with” one or more other components indicatesthat such components are directly connected to each other, in directphysical contact with each other without being connected or attached toeach other, or are not directly connected to each other or in contactwith each other, but are mechanically, electrically (including viaelectromagnetic signals transmitted through space), or fluidicallyinterconnected so as to cause or enable the components so associated toperform their intended functionality.

Cooling may also be provided by a closed loop water jacket coolingsystem, a cooling system mounted on the reactor, or by standard heatexchange through a cover/jacket on the reusable support structure (e.g.,the heat blanket or a packaged dual unit which provides heating andcooling may a component of a device configured for both heating/coolingbut may also be separate from a cooling jacket). Cooling may also beprovided by Peltier coolers. For example, a Peltier cooler may beapplied to an exhaust line to condense gas in the exhaust air to helpprevent an exhaust filter from wetting out.

In certain embodiments, a reactor system includes gas cooling forcooling the head space and/or exit exhaust. For example, jacket cooling,electrothermal and/or chemical cooling, or a heat exchanger may beprovided at an exit air line and/or in the head space of the container.This cooling can enhance condensate return to the container, which canreduce exit air filter plugging and fouling. In some embodiments,purging of pre-cooled gas into the head space can lower the dew pointand/or reduce water vapor burden of the exit air gas.

In some cases, sensors and/or probes (e.g., probe 106) may be connectedto a sensor electronics module 132, the output of which can be sent to aterminal board 130 and/or a relay box 128. The results of the sensingoperations may be input into a computer-implemented control system 115(e.g., a computer) for calculation and control of various parameters(e.g., temperature and weight/volume measurements) and for display anduser interface. Such a control system may also include a combination ofelectronic, mechanical, and/or pneumatic systems to control heat, air,and/or liquid delivered to or withdrawn from the disposable container asrequired to stabilize or control the environmental parameters of theprocess operation. It should be appreciated that the control system mayperform other functions and the invention is not limited to having anyparticular function or set of functions.

The one or more control systems can be implemented in numerous ways,such as with dedicated hardware and/or firmware, using a processor thatis programmed using microcode or software to perform the functionsrecited above or any suitable combination of the foregoing. A controlsystem may control one or more operations of a single reactor for abiological, biochemical or chemical reaction, or of multiple (separateor interconnected) reactors.

Each of systems described herein (e.g., with reference to FIG. 3), andcomponents thereof, may be implemented using any of a variety oftechnologies, including software (e.g., C, C#, C++, Java, or acombination thereof), hardware (e.g., one or more application-specificintegrated circuits), firmware (e.g., electrically-programmed memory) orany combination thereof.

Various embodiments according to the invention may be implemented on oneor more computer systems. These computer systems, may be, for example,general-purpose computers such as those based on Intel PENTIUM-type andXScale-type processors, Motorola PowerPC, Motorola DragonBall, IBM HPC,Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety ofprocessors available from Advanced Micro Devices (AMD) or any other typeof processor. It should be appreciated that one or more of any type ofcomputer system may be used to implement various embodiments of theinvention. The computer system may include specially-programmed,special-purpose hardware, for example, an application-specificintegrated circuit (ASIC). Aspects of the invention may be implementedin software, hardware or firmware, or any combination thereof. Further,such methods, acts, systems, system elements and components thereof maybe implemented as part of the computer system described above or as anindependent component.

In one embodiment, a control system operatively associated with a vesseldescribed herein is portable. The control system may include, forexample, all or many of the necessary controls and functions required toperform a fluidic manipulation (e.g., mixing and reactions) in thecontrol system. The control system may include a support and wheels forfacilitating transport of the vessel. Advantageously, such a portablecontrol system can be programmed with set instructions, if desired,transported (optionally with the vessel), and hooked up to a vessel,ready to perform a fluid manipulation in a shorter amount of time thanconventional fluid manipulation control systems (e.g., less than 1 week,3 days, 1 day, 12 hours, 6 hours, 3 hours, or even less than 1 hour).

A vessel, including the container, may also be connected to one or moresources of gases such as air, oxygen, carbon dioxide, nitrogen, ammonia,or mixtures thereof, in various aspects of the invention. The gases maybe compressed, pumped, etc. Such gases may be used to provide suitablegrowth and/or reaction conditions for producing a product inside thecontainer. The gases may also be used to provide sparging to thecontents inside the container, e.g., for mixing or other purposes. Forinstance, in certain embodiments employing spargers, bubble size anddistribution can be controlled by passing an inlet gas stream through aporous surface prior to being added to the container. Additionally, thesparging surface may be used as a cell separation device by alternatingpressurization and depressurization (or application of vacuum) on theexterior surface of the porous surface, or by any other suitable method.

As a specific example, FIG. 3 shows various sources of gases 118 and124. The inlet gases may optionally pass through filter 120 and/or aflow meter and/or valve 122, which may be controlled by controllersystem 115, prior to entering the container. Valve 122 may be apneumatic actuator (actuated by, e.g., compressed air/carbon dioxide orother gas 124), which may be controlled by a solenoid valve 126. Thesesolenoid valves may be controlled by a relay 128 connected to terminalboard 130, which is connected to the controller system 115. The terminalboard may comprise, for example, a PCI terminal board, or aUSB/parallel, or fire port terminal board of connection. In otherembodiments, flush closing valves can be used for addition ports,harvest and sampling valves. Progressive tubing pinch valves that areable to meter flow accurately can also be used. In some cases, thevalves may be flush closing valves (e.g., for inlet ports, outlet ports,sampling ports, etc.). The inlet gases may be connected to any suitableinlet of the vessel. In one embodiment, the inlet gases are associatedwith one or more spargers which can be controlled independently, asdescribed in more detail below.

As shown in the exemplary embodiment illustrated in FIG. 3, thecontainer and support structure illustrated in FIG. 1 can be operativelyassociated with a variety of components as part of an overall bioreactorsystem 100, according to another aspect of the invention. Accordingly,the container and/or support structure may include several fittings tofacilitate connection to functional component such as filters, sensors,and mixers, as well as connections to lines for providing reagents suchas liquid media, gases, and the like. The container and the fittings maybe sterilized prior to use so as to provide a “sterile envelope”protecting the contents inside the container from airborne contaminantsoutside. In some embodiments, the contents inside the container do notcontact the reusable support structure and, therefore, the reusablesupport structure can be reused after carrying out a particularchemical, biochemical and/or biological reaction without beingsterilized, while the container and/or fittings connected to thecontainer can be discarded. In other embodiments, the container,fittings, and/or reusable support structure may be reused (e.g., aftercleaning and sterilization).

A vessel may also include a mixing system for mixing contents of thecontainer, in another aspect. In some cases, more than one agitator ormixer may be used, and the agitators and/or mixes may the same ordifferent. More than one agitation system may be used, for example, toincrease mixing power. In some cases, the agitator may be one in whichthe height can be adjusted, e.g., such that the draft shaft allowsraising of an impeller or agitator above the bottom of the tank and/orallows for multiple impellers or agitators to be used. A mixing systemof a vessel may be disposable or intended for a single use (e.g., alongwith the container), in some cases.

Various methods for mixing fluids can be implemented in the container.For instance, mixers based on magnetic actuation, sparging, and/orair-lift can be used. Direct shaft-drive mixers that are sealed and notmagnetically coupled can also be used. In one particular embodiment,mixing systems such as the ones disclosed in U.S. patent applicationSer. No. 11/147,124, filed Jun. 6, 2005, entitled “Disposable BioreactorSystems and Methods,” by G. Hodge, et al., published as U.S. PatentApplication Publication No. 2005/0272146 on Dec. 8, 2005, which isincorporated herein by reference in its entirety, are used withembodiments described herein. For example, the mixing system may includea motor 112, e.g., for driving an impeller (or other component used formixing) positioned inside the container, a power conditioner 114, and/ora motor controller 116.

In some cases, a plurality (e.g., more than 1, 2, or 3, etc.) of mixersor impellers are used for mixing contents in a container. Additionallyand/or alternatively, a mixing system may include an adjustable heightimpeller and/or an impeller with varying impeller blade configurations.For instance, the mixer may have an extended drive shaft which allowsthe impeller to be raised to different heights relative to the bottom ofthe container. The extended shaft can also allow integration of multipleimpellers. In another embodiment, a bioreactor system includes more thanone agitation drive per container, which can increase mixing power.

To enhance mixing efficiency, the container may include baffles such asinternal film webs or protrusions, e.g., positioned across the inside ofthe container or extending from the inner surface of the container atdifferent heights and at various angles. The baffles may be formed of inany suitable material such as a polymer, a metal, or a ceramic so longas they can be integrated with the container.

In one embodiment, a direct drive agitator is used. Typically, theagitator includes a direct shaft drive that is inserted into thecontainer. In certain instances, the location where the shaft exits thecontainer may be maintained in a sterile condition. For instance,internal and/or external rotating seals may be used to maintain asterile seal, and/or live hot steam may be used to facilitatemaintenance of the sterile seal. By maintaining such a sterile seal,contamination caused by the shaft, e.g., from the external environment,from the exiting gases, etc., may be reduced or avoided.

In another embodiment, a magnetic agitator is used. Typically, amagnetic agitator uses magnets such as fixed or permanent magnets torotate or otherwise move the agitator, for example, impellers, blades,vanes, plates, cones, etc. In some cases, the magnets within themagnetic agitator are stationary and can be turned on or activated insequence to accelerate or decelerate the agitator, e.g., via an innermagnetic impeller hub. As there is no penetration of the container by ashaft, there may be no need to maintain the agitator in a sterilecondition, e.g., using internal and/or external rotating seals, live hotsteam, or the like.

In yet another embodiment, an electromechanical polymeric agitator isused, e.g., an agitator that includes an electromechanical polymer-basedimpeller that spins itself by “paddling,” i.e., where the agitator ismechanically flapped to propel the agitator or impeller, e.g.,rotationally.

Specific non-limiting examples of devices that can be used as a mixingsystem, and/or an antifoaming system in certain embodiments, areillustrated in FIGS. 4A-8. The devices shown include amagnetically-actuated impeller, although other arrangements arepossible. In some of these magnetic configurations, the motor is notdirectly connected to the impeller. Magnets associated with a drive headcan be aligned with magnets associated with an impeller hub, such thatthe drive head can rotate the impeller through magnetic interactions. Insome cases, the motor portion (and other motor associated components)may be mounted on the support structure.

As shown in FIG. 4A, this exemplary system generally includes animpeller support 300 affixed to portions of a container wall 302,preferably at a lower portion thereof, an impeller hub 304, a motor 306,a motor shaft 308 and a drive head 310. The impeller support may beaffixed to the wall of the container using any suitable technique, e.g.,by heat welding together two portions of a two-piece impeller support,sandwiching the container wall therebetween or onto the wall, or usingother methods described herein. As one example, an opening in the wallof the container may be used to allow a central portion of the impellerplate to extend from an exterior of the container to the interior (orvise versa). Then a sealing ring (not shown) may be adhered or thecontainer may be welded directly to an outer circumference of theimpeller support to seal the container wall therebetween. As anotherexample, an undersized opening in the wall of the container may be usedto form a seal with a circumferencial edge of the impeller supportslightly larger than the opening. In other embodiments, at least aportion of the impeller support is embedded with a wall of the containerand/or the impeller support and container are fabricated simultaneously(e.g., by spin casting, injection molding, or blow molding).

One feature according to one embodiment of the invention is directed tothe inclusion of one or more spargers associated with an impellersupport, which may be used to direct air or other gases into thecontainer. In some cases, the sparger may include porous, micro-porous,or ultrafiltration elements 301 (e.g., sparging elements). The spargersmay be used to allow a gaseous sparge or fluids into and/or out of thecontainer by being dimensioned for connection to a source of a gas; thisconnection may take place via tubing 306. Such sparging and/or fluidaddition or removal may be used, in some cases, in conjunction with amixing system (e.g., the rotation of the impeller hub). Sparging systemsare described in more detail below.

In the embodiment illustrated in FIG. 4A, the interior side of theimpeller support may include a shaft or post 312 to which a centralopening in the impeller hub 304 receives. The impeller hub may bemaintained at a slight distance 305 above the surface of the impellersupport (e.g., using a physical spacer) to prevent frictiontherebetween. Low friction materials may be used in the manufacture ofthe impeller hub to minimize friction between the impeller hub and thepost. In another embodiment, one or more bearings may be included toreduce friction. For instance, the impeller hub may include, in certaininstances, a bearing 323 (e.g., a roller bearing, ball bearing (e.g., aradial axis ball bearing), thrust bearing, race bearing, double racewaybearing, lazy-susan bearing, or any other suitable bearing) for reducingor preventing friction between the impeller support and the post.Additionally, the drive head may include a physical spacer 324 forreducing or preventing friction between the drive head and the impellersupport.

The impeller hub also may include one or more magnets 314, which may bepositioned at a periphery of the hub or any other suitable position, andmay correspond to a position of a magnet(s) 316 provided on the drivehead 310. The poles of the magnets may be aligned in a manner thatincreases the amount of magnetic attraction between the magnets of theimpeller hub and those of the drive head.

The drive head 310 may be centrally mounted on a shaft 308 of motor 306.The impeller hub also may include one or more impeller blades 318. Insome cases, the embedded magnet(s) in the impeller can also be used toremove ferrous or magnetic particles from solutions, slurries, orpowders.

An example of such a system is described in more detail in U.S. patentapplication Ser. No. 11/147,124, filed Jun. 6, 2005, entitled“Disposable Bioreactor Systems and Methods,” by G. Hodge, et al.,published as U.S. Patent Application Publication No. 2005/0272146 onDec. 8, 2005, incorporated herein by reference.

FIG. 4B illustrates another embodiment, having a mechanically-drivenimpeller. As shown, this embodiment generally includes an impellersupport 400, an impeller hub 404 with shaft 405, and an external motor406 with shaft 408. The connection of shafts between the impeller hubshaft and the motor shaft may be accomplished in a matter familiar toone of ordinary skill in the art (e.g., gear box, hex drive, or thelike).

The impeller support can be affixed, for instance, to a side of thebioreactor wall 402 at a lower portion thereof. The impeller support maybe affixed to the wall of the bioreactor by any of the methods discussedherein. Porous, micro-porous, or ultrafiltration elements 401 may alsobe included in the present embodiment to allow gaseous sparge or fluidsinto and out of the bioreactor, as discussed in detail below. In theembodiment illustrated in FIG. 4B, the shaft of the impeller hub may bereceived in a seal 412 (which may also include a bearing, in some cases)centrally located in an impeller support 400. The seal can be used toinsure that the contents of the container are not contaminated. Theimpeller hub can also be maintained at a slight distance above thesurface of the impeller support to prevent friction therebetween. Theimpeller hub may include one or more impeller blades 418, or othersuitable mixing structures, such as vanes, plates, cones, etc.

One aspect of the invention involves the recognition that a careful andclose alignment, vertically and horizontally, between the drive head andimpeller support can add significant benefits to devices of theinvention. Prior to the invention, this may not have been recognized orappreciated as indicated by the general acceptance of the utility, andpotential lack of need for improvement, of typical magneticstirring/rotating mechanisms for fluids. An example includes traditionalmagnetic stir bar arrangements in chemical and biochemical laboratories,where it is typically quite acceptable for magnetic stirrer motors, andstir bars located within a reaction flask, to be not carefully alignedor carefully positioned in proximity to each other. The presentinvention involves recognition that better positioning and proximity canbe achieved through techniques of the invention and result in betterpotential stirring torque and/or stirring rotational rates.

Referring now to FIG. 5, one embodiment of a drive head magneticallycoupled to an impeller of the invention is illustrated schematically. InFIG. 5, an impeller support 501, shown in a cross-section, includes asubstantially horizontal portion 504, from which a substantiallyvertical impeller shaft 508 extends upwardly supporting an impeller 509(which may include a core 510 and blades 511). Impeller 509 may rotateabout shaft 508. Optionally, this rotation may be facilitated by abearing 507, which may be any suitable bearing such as a roller bearing,ball bearing (e.g., a radial axis ball bearing), thrust bearing, racebearing, double raceway bearing, lazy-susan bearing, or the like.Impeller support 501 includes drive head alignment elements 512 which,in the embodiment illustrated, are substantially verticaldownwardly-depending ridges which can define a circular recess intowhich at least a portion of a drive head 516 can be inserted. Guideelements 512 are positioned such that drive head, when engaged with theimpeller support, position the drive head at a predetermined desiredlocation relative impeller 509. In one arrangement, guide elements 512center the drive head, when engaged with the impeller support, withrespect to impeller 509. As a further, optional embodiment, a physicalspacer 520 can be provided between drive head 516 and a bottom surface524 of the impeller support aligned with that portion of the top surface526 of the drive head at the location at which the drive head is ideallypositioned with respect to the impeller support. Physical spacer 520physically separates, by a desired distance, the bottom surface 524 ofthe impeller support with a top surface 526 of the drive head, but, atleast one portion between the top surface of the drive head and bottomsurface of the impeller support, may define a continuous, physicalconnection (free of voids of air or the like), between the drive headand the impeller support. This allows for closer tolerance of the drivehead with the impeller support than would have been realized in manyprior arrangements, and it allows for reproducible and secure engagementof the drive head with the impeller support. In some cases, the drivehead includes a recess 528 into which at least a portion of physicalspacer 520 can be inserted. This arrangement can allow reproducible andsecure engagement of the drive head with the physical spacer.

The bottom of the impeller support and the top surface of the drive headcan be separated (e.g., using a physical spacer) by a distance 521. Inone embodiment, distance 521 is no greater than 50% of average thickness530 of the substantially horizontal portion 504 of the impeller support.In other embodiments, this distance is no more than 40%, 30%, 20%, 10%,or 5% of the thickness of the impeller support.

In some embodiments, physical spacer 520 has a thickness no greater than50% of average thickness 530 of the substantially horizontal portion 504of the impeller support. In other embodiments, this thickness is no morethan 40%, 30%, 20%, 10%, or 5% of the thickness of the impeller support.

In one set of embodiments, physical spacer 520 is a bearing thatfacilitates rotation of the drive head relative to the impeller support.Where physical spacer 520 is a bearing, any suitable bearing can beselected such as a roller bearing, ball bearing (e.g., a radial axisball bearing), thrust bearing, race bearing, double raceway bearing,lazy-susan bearing, or the like.

In the embodiment illustrated in FIG. 5, the drive head can vary inposition, relative to shaft 508, horizontally no more than 5 mm duringnormal operation or, in other embodiments, no more than 4, 3, 2, 1 (0.5,or 0.25 mm during normal operation). The drive head can also vary indistance relative to bottom surface 524 of the impeller support by nomore than 10 mm, 1 mm, 0.5 mm, 0.25 mm, 0.1 mm, or 0.005 mm in certainembodiments with the use of the arrangements illustrated in FIG. 5.

The arrangements of FIG. 5, especially in embodiments where physicalspacer 520 is used, also adds physical support to impeller support 501in addition to any other physical support which the impeller support 501might receive. This added support is particularly advantageous incollapsible bag arrangements including impellers (e.g., for mixersand/or antifoaming devices).

Optionally, impeller support 501 may include spargers 540 positionedbeneath blades of the impeller. The spargers can be dimensioned forconnection to one or more sources of gas. For example, the spargers mayinclude a port that can be connected to tubing 542 in fluidcommunication with one or more sources of gas.

Although many of the figures described herein show impellers that arepositioned at or near a bottom portion of a container, in otherembodiments, impellers can be positioned at any suitable location withina container, for example, near the center or a top portion of acontainer. This can be achieved by extending the length of a shaft whichsupports the impeller, or by any other suitable configuration. Positionsof impellers in a container may depend on the process to be performed inthe container. For instance, in some embodiments where sparging isrequired, impellers may be positioned near the sparger such that theimpeller can sweep and/or regulate the bubbles introduced into thecontainer. Additionally, although the figures described herein show asingle impeller associated with a shaft, more than one impeller can beused in some instances. For example, a first impeller coupled to a shaftmay be located near a bottom portion of the container and a secondimpeller coupled to the shaft may be positioned near the center of thecontainer. The first impeller may provide adequate sweeping of a spargedgas, and the second impeller may provide adequate mixing of contentswithin the container.

In one aspect of the invention, the impeller support is uniquelydesigned to be readily fastenable to a collapsible bag. Certain knowarrangements of impellers attached to collapsible bags may suffer fromdrawbacks resulting from non-ideal attachment of the bag to the impellersupport, or non-ideal techniques for such attachment, or both. As shownin the embodiment illustrated in FIG. 5, the present invention, in thisaspect, includes an impeller support having a base, substantiallyperpendicular to a shaft upon which the impeller rotates, having a firstportion 534 of average thickness sufficient to adequately support theimpeller shaft, and a second, peripheral portion 536 thinner than thefirst portion for facilitating attachment to the bag. The first portionthickness is defined as the overall thickness cross-section taken up bythe first portion at any point and, where the first portion includes aribbed or other structure including various thicknesses, the thicknessfor purposes of this discussion is defined as the thickest portion. Thesecond, peripheral portion, in one embodiment, defines a compositionsimilar to or essentially identical to that of the collapsible bag, andis provided in a thickness similar to that of the collapsible bag. Inother embodiments, the second, peripheral portion is formed by acomposition different than that of the collapsible bag. For instance, insome embodiments, the first portion is formed in low densitypolyethylene, and the second portion is formed in high densitypolyethylene, polypropylene, silicone, polycarbonate, and/orpolymethacrylate.

The thickness of the peripheral portion of the support and the thicknessof the walls of collapsible bag 540, prior to attachment, may differ byno more than 100%, or by no more than 80%, 60%, 40%, 20%, or 10% inother embodiments (e.g., as a percentage of the greater thicknessbetween the walls of the bag and the peripheral portion). This aspect ofthe invention involves, in part, the discovery that where the thicknessof the peripheral portion of the impeller support and the thickness ofthe disposable bag (at least the portion attachable to the impellersupport) are made of similar (or compatible) materials and are ofsimilar thickness, then joining of one to the other can be facilitatedeasily, reproducibly, and with a product that is free of significantirregularity and thickness in the transition of the bag to the impellersupport attachment portion. Thus, one aspect of the invention involvesthe product of attachment of a collapsible bag and an impeller supporteach as defined above, and in another aspect involves a kit including animpeller support and a collapsible bag prior to attachment. As describedherein, joining of the bag and the support can be performed by anysuitable method including, for example, molding (e.g., as describedabove in connection with FIGS. 2A-2C) and welding (e.g., ultrasonic orheat welding).

Another aspect of the invention involves impellers with replaceableblades. FIG. 6 illustrates an impeller 570 according to one embodimentof this aspect of the invention which includes a hub 572 which can havea generally circular outer perimeter and may include a center passage576 before within which the impeller shaft or post (not illustrated)resides. Hub 572 includes one or more slots 578 within which one or moreimpeller blades 580 can, in some embodiments, be replaceably inserted.As illustrated, one slot 578 is shown not containing a blade and oneslot 578 is shown containing an impeller blade. The blade and bladeslots are illustrated very schematically and, of course, those ofordinary skill in the art will recognize that a variety of differentsizes, shapes, and pitches of blades and slots can be selected by thoseof ordinary skill in the art for a variety of mixing purposes describedherein and known in the art. Blades 580 can be positioned and heldwithin slots 578 securely enough for suitable use and accordance withthe invention by any number of techniques including, for example,friction fitting, press fitting, detent mechanism, a clipping and cliprelease arrangement, fastening with screws, pegs, clamps, or the like,welding (e.g., heat and ultrasonic welding), and use of adhesives.

The replaceable blade arrangement of the invention as illustrated inFIG. 6 provides the advantage in that different blades can be used witha single hub in a mixing/rotating arrangement so that the arrangementcan be used for different purposes or involving different rotationalspeed, torque, mixing profile, or the like. For example, blades of afirst size or pitch can be replaced with blades of a second size orpitch to create greater or lesser sheer, aeration, mixing or the like aswould be understood by those of ordinary skill in the art. Whilereplaceable blades (e.g., airplane propeller blades) are known indifferent fields, replaceable blades in a collapsible bag arrangementsuch as that of the present invention would not have been expected tohave been found based upon knowledge in the art because such bagstypically were used only for mixing media containing cells which, toavoid being lysed, must be stirred below a threshold of sheer, or formedia containing other materials which can tolerate much higher sheer.In this aspect of the present invention, collapsible bag arrangementscan be prepared with multiple blades and provided for use with either orboth of two or more mixing profiles.

In one aspect, the impeller (in some embodiments, via magnetic couplingof the drive head to the impeller) is driven by a motor able to reverseits direction of rotation and/or to be finely tuned with respect torotational speed. Reversal of direction of spin is not found orsuggested by the known art but provides significant advantage asrecognized by the present invention in terms of variety ofaeration/sparger profiles, or the like. Fine tuning of impeller speedhas been determined according to the present invention to allow for aprecise and controllable degree and/or balance of aeration/sparging,sheer, or the like, which has been determined to be quite useful inconnection with various media for mixture, especially those includingcells. This embodiment of the invention allows for reproducible andcontrollable adjustment of rotational speed of the impeller that amountsof plus or minus 5% or less through a range of rotational speeds ofbetween 10% and 90% of total maximum impeller rotational speed. In otherembodiments, rotational tuning of 4%, 3%, 2%, or 1% of this speed isfacilitated. In one arrangement, these aspects are realized by use of aservo motor.

In another aspect, the present invention includes a system that canreduce foam produced within a container or reduce the amount of foamcontained in a head space of a container. In some cases, sensors and/orcontrollers may also be used to monitor and/or control foaming. As aspecific, non-limiting example, a foam sensor may be inserted into ahead space. In one embodiment, the foam sensor may include two or morefoam probes with a voltage potential between them that is able toactivate antifoaming (via a control system), for example, the additionof a chemical antifoaming agent, e.g., via a pump. In anotherembodiment, a foam sensor includes a first portion including a probe anda second portion including a wall of a reusable support structure (or awall of a container) that can detect a change of electrical current flowbetween the probe and the wall (e.g., due to the presence of the foam).In another embodiment, a foam sensor uses an amperage draw of anexternal motor to detect foam in a container. In such an arrangement,the current may increase when the foam contacts an impeller driven bythe motor. In yet another embodiment, the foam sensor can activate amechanical antifoaming device via a control system. As another specificnon-limiting example, mechanical foam control may be achieved, forexample, via a mounting impeller in head of a container, turned upsidedown, optionally with fritted or non-fritted exhaust air exit elementsto enhance foam breakage. Examples of impellers that can be used in anantifoaming system are described above in connection with FIGS. 4A-8.

More generally, in some embodiments, a device able to reduce oreliminate foam may be associated with a vessel, for example, within acontainer of the vessel. The antifoaming device may be able to reduce oreliminate foaming, for example, prior to foam accumulating in an exhaustor an outlet, plugging of filters, or the like. The antifoaming systemmay be run continuously, periodically, or in some cases, in response tocertain events, e.g., within a bioreactor system and/or within thecontainer. For example, the antifoaming device may include one or moresensors and a control system which is able to monitor foaming and act toreduce or eliminate the foaming.

In various embodiments, the antifoaming device may include a controlsystem, which can include one or more foam sensors, for example, withina container. The foam sensor may be positioned in any place able todetect foaming and/or a consequence of foaming, for example, thealteration of pressure within an outlet of the container. For instance,a foam sensor may be positioned in a head space within the container, inan outlet port, in an exit line (e.g., in an exit air line, and/orupstream of an exit air filter), or the like. Detection of foam in oneor more of the foam sensors may cause the control system to enactantifoam measures, as discussed below. In another set of embodiments, asensor that detects foam or the effects of foam in a container may be apressure sensor that detects overpressure (e.g., due to clogging by thefoam), which may be used to determine the degree of foaming. Forexample, in one embodiment, foam detection may occur by a pressuresensor in a head space within a container, or within an outlet port.

Antifoaming techniques include, but are not limited to, a lowering ofgas flow rate to the container and/or shut of gassing entirely, theaddition of chemical antifoaming agents, which are known to those ofordinary skill in the art (e.g., via an external pump), a mechanicalfoam breaker mounted in head space, and/or the lowering or ceasing of anagitation rate of a mixer in the container.

In one set of embodiments, a mechanical antifoaming system is used toreduce or eliminate foaming. The mechanical antifoaming system can bepositioned in any suitable location within the bioreactor system such aswithin the container. For instance, a mechanical antifoaming system maybe fitted to the inside head space of a container of the presentinvention, or to an outlet port (which allows air and/or foam to exitthe container). The mechanical antifoaming system may have any suitablestructure able to reduce and/or eliminate foam. For instance, in oneembodiment, the mechanical foam breaker includes one or more stainlesssteel plates or cones mounted on a hollow rotating shaft that penetratesthe container. The shaft can be rotated by an external motor (e.g., amagnetically-operated motor) or other suitable apparatus. In some cases,a hollow motor shaft may be used, which may also provide a passage forgases exiting the container in some cases. In other cases, impellerssuch as the ones shown in FIGS. 4A-8 can be used.

In certain instances, a shaft associated with a mechanical antifoamingdevice is positioned between the inside and outside of the container.The location where the shaft exits the container may be maintained in asterile condition. For instance, internal and/or external rotating sealsmay be used to maintain a sterile seal, and/or live hot steam may beused to facilitate maintenance of the sterile seal. By maintaining sucha sterile seal, contamination caused by the shaft, e.g., from theexternal environment, from the exiting gases, etc., may be reduced oravoided.

Without wishing to be bound by any theory, it is believed that, as gasand/or foam passes through a mechanical antifoaming device, for example,between spinning impellers, plates or cones, a centrifugal force isapplied to the foam which overcomes stabilizing surface tension forcesand can result in collapse of the foam bubble. The fluid from thecollapsed bubble may thus be ejected out and down to the container.Thus, the exiting gases may be at least substantially free of foam. Themechanical antifoaming device can be left running at all times oractivated as needed via a sensor, e.g., via a sensor mounted in the headspace of the container, as previously described.

Referring now to FIG. 7, one embodiment of such a mechanical antifoamingsystem is shown. In this figure, a foam 602 is produced, e.g., during achemical, biochemical, and/or a biological reaction within a container608 (e.g., a rigid container or a collapsible bag). In this example,foaming is detected by a foaming sensor 611 in electrical communicationwith a control system, which then causes an antifoaming device toactivate. Here, the antifoaming device includes an external drive motor614, which rotates a shaft 617 connected to vanes 622. Shaft 617penetrates container 608 to reach its interior. The foam 602 is thendisrupted as it is passed between spinning vanes 622. In addition, inthis example, shaft 617 is hollow, and gases can pass through the shaft,exiting container 608, as indicated by arrows 619, passing throughconduits 616 and optional filter 617.

In another embodiment, a mechanical antifoaming device of the inventiondoes not penetrate the container. Thus, the mechanical antifoamingdevice may lack a rotating shaft or a hollow shaft, and/or may lack aseal. In addition, as there is no penetration of the container by such amechanical antifoaming device, there may be no need to maintain themechanical antifoaming device in a sterile condition, e.g., usinginternal and/or external rotating seals, live hot steam, or the like.Such a system may be formed, for instance, from steel, stainless steelor inexpensive hard plastic for installation in collapsible ordisposable bags or other containers described herein.

Referring now to FIG. 8, a non-limiting example of such a mechanicalantifoaming system is shown. In this figure, within container 608, foam602 is produced, e.g., during a chemical and/or a biological reactionwithin the container. The foaming is detected by a foaming sensor 611,and a control system then causes an antifoaming device to activate. Inthis particular example, the antifoaming device includes an externaldrive motor 614, which includes a spinning external magnetic drive head630. The lack of a penetrating shaft allows container 608 to be quicklyremoved from motor 614. Inside container 608, an internal magnetic hub632 rotates as a result of the rotation of external magnetic drive head632. This causes rotation of blades 638 attached to internal magnetichub 632 (e.g., about a single axis), which then breaks down the foam. Inthis example, gases do not leave via a hollow shaft (as in FIG. 7), butinstead pass through an outlet port 240 (which may be hollow, contain aporous frit element, etc.), exiting container 608, as indicated byarrows 619, passing through conduits 616 and filter 617.

Although FIGS. 7 and 8 show mechanical antifoaming devices that arepositioned at or near a top portion of a container, in otherembodiments, the devices can be positioned at any suitable locationwithin a container, for example, near the center portion of a container.This can be achieved, for example, by extending the length of a shaftwhich supports an impeller, or by any other suitable configuration. Theimpeller can also be lowered or risen depending on the levels of liquidand foam in the container. Additionally, antifoaming devices may includemore than one impeller in some instances. For example, a first impellercoupled to a shaft may be located near a top portion of the containerand a second impeller coupled to the shaft may be positioned near thecenter of container. An antifoaming system of a vessel may be disposableor intended for a single use (e.g., along with the container), in somecases.

The impeller systems described herein may allow the system to mixfluids, solids, or foams of any type. For example, fluids inside thecontainer may be mixed to provide distribution of nutrients anddissolved gases for cell growth applications. The same disposablecontainer may be used for mixing buffers and media or other solutions inwhich a disposable product contact surface is desirable. This may alsoinclude applications in which the vessel is not required to be sterileor maintain sterility. Moreover, embodiments described herein enable thecontainer holding the fluids/mixtures/gases to be removed and discardedfrom the reusable support structure such that the reusable supportstructure is not soiled by the fluids that are mixed in the container.Thus, the reusable support structure need not to be cleaned orsterilized after every use.

Another aspect of the invention includes multiple spargers (includingsparging elements) that may be dimensioned for connection to differentsources of gas and/or which may be independently controlled. The type ofgas, number of spargers, and types and configurations of spargers usedin a bioreactor system or a biochemical/chemical reaction system maydepend, in part, on the particular process to be carried out (e.g., anaerobic versus anaerobic reaction), the removal of any toxic byproductsfrom the liquid, the control of pH of a reaction, etc. As described inmore detail below in connection with certain embodiments describedherein, a system may include separate spargers for different gases whichmay have different functions in carrying out, for example, a chemical,biochemical and/or biological reaction. For instance, a bioreactorsystem for cell cultivation may include different types of gases such asa “dissolved oxygen (DO) control gas” for controlling the amount ofdissolved oxygen in the culture fluid, a “strip gas” for controlling theamount of toxic byproducts in the culture fluid, and a “pH control gas”for controlling the pH of the culture fluid. Each type of gas may beintroduced into the culture using different spargers that can beindependently operated and controlled. Advantageously, such a system mayprovide faster process control and less process control variability(compared to, for example, certain systems that combine a DO controlgas, strip gas, and pH control gas into one gas stream introduced into areactor). Chemical, biochemical and/or biological reactions carried outin bioreactor systems described herein may also require lowerconsumption of gas which can save money on expensive gases, and/or lesstotal gas flow rate (e.g., for a strip gas), which can reduce foamgeneration and/or reduce the size of inlet gas sterile filters required.

In some embodiments, vessels described herein are a part of a bioreactorsystem. In bioreactors used for certain types of cell cultivation, cellsmay require nutrients such as sugars, a nitrogen source (such as ammonia(NH₃) or amino acids), various salts, trace metals and oxygen to growand divide. Like the other nutrients, even and uniform distribution ofoxygen throughout the reactor may be essential to provide uniform cellgrowth. Poor distribution of oxygen can create pockets of cells deprivedof oxygen, leading to slower growth, alteration of the cell metabolismor even cell death. In certain applications where the cells areengineered to produce a bioproduct, oxygen deprivation can have a severaffect on the quantity and quality of bioproduct formation. The amountof nutrients available to cells at any one time depends in part on thenutrient concentration in the fluid. Sugars, nitrogen sources, salts,and trace metals may be soluble in fluid and, therefore, may be inexcess and readily available to the cells. Oxygen, on the other hand,may be relatively poorly soluble or “dissolved” in water. In addition,the presence of salts plus the elevated temperature necessary to growcells may further reduce dissolved oxygen concentration. To compensate,a rapid dissolved oxygen sensing system, constant and steady transfer ofoxygen into the fluid (e.g., using one or more spargers as describedherein), combined with rapid and even distribution in the bioreactor maybe used to reduce or prevent oxygen starvation.

Since oxygen transfer from the gas bubbles entering the fluid of theculture may be an important control parameter, the time constant ofresponsiveness of the gas delivery system may also be important. Incertain embodiments, as cell population density increases, the responserate of the gassing system to supply oxygen enriched DO control gas maybecome increasingly important. Accordingly, in some embodiments, systemsdescribed herein include one or more sensors such as a DO sensor whichdetects the need for more oxygen (or other gas), a gas controller, andone or more spargers which can be signaled to enrich the culture withextra oxygen using, for example, a N₂/O₂/air control gas. Since delaytime (e.g., several minutes) for this enriched gas to reach the reactorcan result in a drop in DO which can lead to oxygen starvation, systemsdescribed herein may include a control feedback loop between thesensor(s), gas controller, and sparger(s). Thus, responsive, and evensupply and distribution of oxygen-bearing control gas (e.g., a N₂/O₂/airmix) may be provided for controlled, predictable cell growth andbioproduct formation. Systems described herein allowing independentcontrol of spargers and/or gas compositions may be advantageous comparedto systems that require gases to be flushed out before sparging adifferent gas into the container.

In addition, since compressed air and oxygen may be expensive to supplyto the reactor, a system that provides just enough air enriched withjust enough oxygen such that the bubbles are not lost to the head spaceof the container (and lost out through the exhaust line) may beimplemented. This can be performed, for example, by controlling theamount and flow rate of a control gas independently of other gases usedin the system (e.g., a strip gas and/or a pH control gas).

Without wishing to be bound by any theory, it is believed that the rateof oxygen transfer into the bioreactor fluid from air, pure oxygen or agas mixture is directly related to the amount of total surface area ofthe bubbles in the fluid. Hence, larger bubbles provide less totalsurface area than a fine mist of very small bubbles. For this reason, incertain embodiments of the invention, a control gas may be providedthrough microporous spargers to create very small bubbles. A microporoussparger may include apertures having a size (e.g., average diameter) of,for example, less than less than 500 microns, less than 200 microns,less than 100 microns, less than 60 microns, less than 50 microns, lessthan 40 microns, less than 30 microns, less than 20 microns, less than10 microns, less than 3 microns, less than about 1 micron, or less than0.1 microns. In certain embodiments, microporous spargers have anaperture size between 0.1 and 100 microns. Of course, spargers havinglarger aperture sizes may also be used. For instance, a sparger may havean aperture size between 0.1 and 10 mm. The aperture size may be greaterthan 100 microns, greater than 200 microns, greater than 500 microns,greater than 1 mm, greater than 3 mm, greater than 5 mm, greater than 7mm, or greater than 10 mm. The aperture may have any suitablecross-sectional shape (e.g., circular, oval, triangular, irregular,square or rectangular, or the like). Spargers having combinations ofaperture sizes can be incorporated into vessels described herein.

Additionally, good cell growth and controlled metabolism may bedependent upon removal of toxic byproducts of cell growth, such as, forexample, carbon dioxide, ammonia and volatile organic acids. Carbondioxide may be highly soluble in water, which can exacerbate its toxiceffect on cells. These byproducts can be “stripped” out of the culturefluid by gassing the culture using a strip gas. Accordingly, evendistribution of strip gas and strip gas that is introduced at a flowrate sufficiently high enough for bubbles to escape out of the culture(and out the exhaust vent, for example) may be important for cell growthand/or bioproduct production. These parameters may be controlledindependently of other gases used in the system (e.g., a control gasand/or a pH control gas) using a separate sparger for the strip gas.

In some instances, a strip gas is introduced into a container using asparger having an aperture size between 0.1 and 10 mm. For example, theaperture size may be greater than 100 microns, greater than 200 microns,greater than 500 microns, greater than 1 mm, greater than 3 mm, greaterthan 5 mm, greater than 7 mm, or greater than 10 mm. These aperturesizes can allow relatively larger bubbles to pass through the liquid ofthe container, which can strip any toxic byproducts out of the liquidwithout creating large amounts of foam in the head space of thecontainer.

In certain embodiments, a pH control gas is used to control the pH ofthe fluid in a bioreactor system. For example, carbon dioxide may beused to increase solution pH and ammonia may be used to decreasesolution pH. In one embodiment, a pH control gas may include acombination of carbon dioxide, ammonia, or other gases to control (e.g.,increase or decrease) pH. In another embodiment, the pH of a reactionfluid is controlled by a first sparger containing an agent thatincreases pH (e.g., CO₂) and a second sparger containing an agent thatdecreases pH (e.g., NH₃).

One or more pH control gases may be added to a container of thebioreactor system upon signals from a pH control sensor associated withthe system. The pH control gases may be operated independently andwithout interference by oxygen demand (e.g., a DO control gas) or stripgas systems. A pH control gas may be introduced into a container usingspargers having apertures of various sizes.

In other embodiments, cells that are normally grown without oxygen(e.g., anaerobic reactions) or which are even sensitive to oxygenrequire removal of oxygen from the culture. Even and controlleddistribution of nitrogen gas in these cultures may be used to controlproper cell growth and product formation.

As mentioned, in some embodiments of the present invention, gases suchas air, CO₂, O₂, N₂, NH₃, and/or dissolved oxygen may be sparged intothe container. In some cases, the sparging can be controlled, forinstance, such that the sparging can be rapidly activated or altered asneeded. Multiple spargers may be used in some cases. For example, in oneembodiment, different gas compositions may each be introduced into thecontainer using multiple spargers, e.g., a first sparger for a first gascomposition, a second sparger for a second gas composition, a thirdsparger for a third gas composition, etc. The gases may differ incomposition and/or in concentration. As a specific example, a first gascomposition may include air with 5% CO₂, and a second gas compositionmay include air with 10% CO₂; in another example, a first gascomposition may include O₂, and a second gas composition may include N₂;in yet another example, a first gas composition may include a controlgas, a second gas composition may include a strip gas, and a third gascomposition may include a pH control gas. Of course, other combinationsof gases are also possible. In some cases, multiple spargers may beuseful to allow faster responses, e.g., as the gas composition beingintroduced into the container may be rapidly changed by activatingdifferent spargers, e.g., singly and/or in combination. As a specificexample, the gas being introduced into a container can be rapidlyswitched from a first gas (via a first sparger) to a second gas (via asecond sparger), and/or to a combination of the first and second gas, ora combination of the second gas and a third gas, etc. The flow rates ofeach gas can also be changed independently of one another. (In contrast,with a single sparger, a change in composition requires that the newcomposition reach the sparger before being introduced into thecontainer.) Moreover, the use of multiple spargers can allowcustomization of the type of sparger for a particular type of gas, e.g.,a strip gas, DO control gas, pH control gas, air, CO₂, O₂, N₂, NH₃, orany other suitable gas, if desired.

Sparging may be run continuously, periodically, or in some cases, inresponse to certain events, e.g., within a bioreactor system and/orwithin the container. For example, as mentioned, the spargers may beconnected to one or more sensors and a control system which is able tomonitor the amount of sparging, the degree of foaming, the amount orconcentration of a substance in the container, and respond byinitiating, reducing, or increasing the degree of sparging of one ormore composition(s) of gases.

In one particular embodiment, a vessel (e.g., as part of a reactorsystem for performing a biological, biochemical or chemical reaction) isconfigured to contain a volume of liquid and includes a container (e.g.,a collapsible bag) having a volume of at least 2 liters (or any othersuitable volume) to contain the volume of the liquid. The vessel mayoptionally include a support structure for surrounding and containingthe container. Additionally, the vessel includes a first spargerconnected or dimensioned to be connected to a source of a first gascomposition in fluid communication with the container, and a secondsparger connected or dimensioned to be connected to a source of a secondgas composition different from the first gas composition in fluidcommunication with the container. The vessel further comprises a controlsystem operatively associated with the first and second spargers andconfigured to operate the spargers independently of each other. Ofcourse, third, fourth, fifth, or greater numbers of spargers can beincluded (e.g., greater than 10, or greater than 20 spargers), dependingon, for example, the size of the container. In some embodiments, thevessel further comprises a mixing system including an impeller and abase plate, wherein the first and/or second spargers is associated withthe base plate. In one particular embodiment, the first gas compositioncomprises air and the second gas composition comprises air supplementedwith O₂ and N₂. If additional spargers are included, the spargers can beconnected to a source of gas comprising N₂, CO₂, NH₃ and/or any othersuitable gas.

In another exemplary embodiment, a vessel configured to contain a volumeof liquid comprises a container (e.g., a collapsible bag) to contain theliquid, and optionally, a support structure for surrounding andcontaining the collapsible bag. The vessel includes a first spargerconnected to the container, the first sparger having a first aperturesize, wherein at least a portion of the first sparger is dimensioned tobe connected to a source of a first gas composition. The vessel alsoincludes a second sparger connected to the container, the second spargerhaving a second aperture size, wherein at least a portion of the secondsparger is dimensioned to be connected to a source of a second gascomposition. The second gas composition may have the same or a differentcomposition than the first gas composition. In some embodiments, thevessel is part of a bioreactor system; or, the vessel may be a part of abiochemical/chemical reaction system, or a mixing system. The vessel mayinclude a control system operatively associated with the first andsecond spargers and may be configured to operate the spargers (or gasesassociated therewith) independently of each other. The vessel mayinclude any suitable number of spargers (e.g., greater than 10 orgreater than 20 spargers), and the container may have any suitablevolume (e.g., at least 2, 10, 20, 40, or 100 liters). The first and/orsecond gas composition(s) may include, for example, N₂, O₂, CO₂, NH₃, orair. For example, in one instance, the first gas comprises air and thesecond gas comprises air supplemented with O₂ and N₂. The first aperturesize may be larger than the second aperture size. For instance, thefirst aperture size may be between 0.1 and 10 mm, and the secondaperture size may be between 0.1 and 100 microns.

Apertures associated with spargers can be formed in any suitablematerial. For instance, in one embodiment, a porous polymeric materialis used as a sparging element to allow transport of gas from one side toanother side of the material. Apertures can also be formed in othermaterials such as metals, ceramics, polymers, and/or combinationsthereof. Materials having pores or apertures can have any suitableconfiguration. For example, the materials may be knitted, woven, or usedto form meshes or other porous elements. The elements may be in the formof sheets, films, and blocks, for example, and may have any suitabledimension. In some cases, such elements are incorporated with impellersor impeller supports, e.g., as illustrated in FIG. 5. The elements canbe positioned and held within regions of the impeller or impellersupport securely enough for suitable use and accordance with theinvention by any number of techniques including, for example, frictionfitting, press fitting, detent mechanism, a clipping and clip releasearrangement, fastening with screws, pegs, clamps, or the like, welding(e.g., heat and ultrasonic welding), and use of adhesives. In otherembodiments, portions of the impeller and/or impeller support can befabricated directly with pores or apertures that can allow fluids toflow therethrough.

The vessel may optionally include one or more sensors in electricalcommunication with the control system for determining an amount orconcentration of a gas (e.g., O₂, N₂, CO₂, NH₃, a bi-product of areaction) in the container. Additionally and/or alternatively, thevessel may include a sensor in electrical communication with the controlsystem for determining a pH of a liquid in the container, or an amountor level of a foam in the bag.

As mentioned, control systems and feedback loops may be used to controlthe degree of sparging in one embodiment, or degree of mixing, oractivity of an antifoaming system in other embodiments. One example ofsuch a control and feedback process is shown in the embodimentillustrated in FIG. 9. System 700 may include a first sensor 702 (e.g.,for detecting the amount and/or concentration of CO₂ of a liquid in thecontainer) and a second sensor 704 (e.g., for detecting the amountand/or concentration of O₂ of a liquid in the container). Aftercalibrating the sensors, reagents may be added to a container 708 and afluidic manipulation process, such as mixing or performing a biological,chemical, or biochemical reaction, may be take place. The amount of agas such as O₂ and CO₂ may vary in the liquid of the container as theprocess proceeds. For example, if a biological reaction involving cellstakes place, the cells may consume O₂ and form CO₂ over time, which mayvary depending on the growth stage of the cells. Thus, the amount and/orconcentration of gases can be determined by the sensors (e.g., as afunction of time), and signals 712 and 714 related to the amounts and/orconcentrations of the gases can be sent to a control system 720. Thecontrol system may include recorded parameters 724, such as thresholdlevels of one or more gases that can inputted by a user prior to orduring the reaction. For example, a parameter may include a certainthreshold level of CO₂ in the liquid before a sparger is activated toreduce the amount of CO₂ using a strip gas. Accordingly, a signal may besent from the control system to activate a component 732, such as avalve connected to a source of a strip gas used to reduce the amount ofCO₂. As the strip gas is introduced into container 708, the amountand/or concentration of CO₂ may decrease, which can be measured by 712and signals sent to the control system. When the amount and/orconcentration of CO₂ decreases to a certain level, the control systemcan lower or deactivate the amount of CO₂ being introduced into thecontainer, thereby completing the feedback loop. A similar process cantake place independently of the process described above using secondsensor 714, which may measure, for example, a second gas, a pH, or anamount of a foam in a head space of the container.

In another aspect, a bubble column or airlift system (utilizing bubblesof air or other gas) may be used with the disposable bioreactor bag.Such a system may provide a mixing force by the addition of gas (e.g.,air) near the bottom of the reactor. Here, the rising gas bubble and thelower density of gas-saturated liquid rise, displacing gas-poor liquidwhich falls, providing top-to-bottom circulation. The path of risingliquid can be guided, for example, using dividers inside the chamber ofthe bag. For instance, using a sheet of plastic which bisects theinterior of the bioreactor bag, e.g., vertically, with a gap at the topand the bottom. Gas may be added on one side of the divider, causing thegas and gas-rich liquid to rise on one side, cross over the top of thebarrier sheet, and descend on the other side, passing under the dividerto return to the gas-addition point. In addition, such a bubblecolumn/air-lift mixing system and method may be combined with any of theother mixing systems described herein.

In one aspect, a bioreactor system as described herein includes anenclosed resin loading/column packing system. Typically, column packingtypically may be accomplished in a clean room with open carboyscontaining the resin which is manually mixed while the resin slurry ispumped onto the column. In one embodiment, however, a container such asa flexible container is loaded with chromatography resin which isslurried by an agitator while the slurry is pumped into a column.

In certain chemical, biochemical and/or biological processes requiringlight, a bioreactor system described herein may include direct, indirectand/or piped-in lighting, e.g., using fiber-optics, according to anotheraspect of the invention. Any suitable light source may be used. Suchbioreactor systems may be useful for processing, for example, plantcells, e.g., to activate photosynthesis. In one particular embodiment, aphosphorescent flexible container is used to provide light, e.g., forgrowth of plant cells.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.” It should also beunderstood that, unless clearly indicated to the contrary, in anymethods claimed herein that include more than one step or act, the orderof the steps or acts of the method is not necessarily limited to theorder in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

1. A vessel configured to contain a volume of liquid, the vesselcomprising: a collapsible bag for containing the volume of liquid, thecollapsible bag having a volume of at least 2 liters; a supportstructure surrounding and containing the collapsible bag; a firstsparger connected to the collapsible bag, the first sparger being influid communication with a source of a first gas composition; and asecond sparger connected to the collapsible bag, the second spargerbeing in fluid communication with a source of a second gas compositiondifferent from the first gas composition. 2-4. (canceled)
 5. A vessel asin claim 1, wherein the vessel further comprises a mixing systemincluding an impeller and a base plate, wherein the first and/or secondspargers is associated with the base plate. 6-17. (canceled)
 18. Avessel configured to contain a volume of liquid, the vessel comprising:a collapsible bag able to contain the volume of liquid; a supportstructure surrounding and containing the collapsible bag; a firstsparger connected to the collapsible bag, the first sparger having afirst aperture size, wherein at least a portion of the first sparger isdimensioned to be connected to a source of a first gas; and a secondsparger connected to the collapsible bag, the second sparger having asecond aperture size, wherein at least a portion of the second spargeris dimensioned to be connected to a source of a second gas. 19.(canceled)
 20. A vessel as in claim 18, wherein the vessel furthercomprises a control system operatively associated with the first andsecond spargers and configured to operate the first and second spargersindependently of each other. 21-29. (canceled)
 30. A vessel configuredto contain a volume of liquid comprising: a container able to containthe volume of liquid; and a magnetically-driven antifoaming device, atleast a portion of which is positioned in a head space of the containerwhen the container contains the volume of liquid, the antifoaming devicebeing configured and arranged to break up foam in the head space duringrotation of at least a portion of the antifoaming device.
 31. A vesselas in claim 30, wherein the container is a collapsible bag.
 32. A vesselas in claim 30, further comprising a reusable support structuresurrounding and containing the collapsible bag. 33-34. (canceled)
 35. Avessel as in claim 34, wherein the impeller is connected to a topportion of the container. 36-46. (canceled)
 47. A vessel as in claim 46,wherein the antifoaming device is regulated by the control system viadetection of electrical current flow between the probe and the wall.48-57. (canceled)
 58. A vessel configured to contain a volume of liquid,the vessel comprising: a collapsible bag able to contain the liquid; areusable support structure surrounding and containing the collapsiblebag; a pressure sensor for determining a pressure in the collapsiblebag, the pressure sensor in fluid communication with the collapsiblebag; an antifoaming device associated with the collapsible bag andconfigured to break up foam in the collapsible bag; and a control systemoperatively associated with the pressure sensor and the antifoamingdevice, wherein the control system regulates the antifoaming device uponreceipt of a signal from the pressure sensor. 59-63. (canceled)
 64. Acollapsible bag configured to contain a volume of at least 2 liters, thecollapsible bag comprising: a first rotatable impeller positioned at abottom portion of the collapsible bag, the first impeller able to bemagnetically rotated; and a second impeller positioned at a top portionof the collapsible bag, the second impeller able to be magneticallyrotated. 65-70. (canceled)
 71. A collapsible bag as in claim 64, whereinthe sparger is positioned below a bottom impeller and an outlet ispositioned above a top impeller.
 72. A container able to contain avolume of liquid, the container comprising: a collapsible bag having avolume of at least 40 liters, wherein the collapsible bag does notinclude any seams joining two or more flexible wall portions of thecollapsible bag; and a reusable support structure surrounding andcontaining the collapsible bag. 73-77. (canceled)
 78. A container as inclaim 72, wherein the collapsible bag comprises an impeller. 79.(canceled)
 80. A method comprising: positioning a rigid component in amold having a shape configured to mold a container having a volume of atleast 10 mL; introducing a first polymer precursor into the mold; andforming a seamless container within the mold by solidifying the polymerprecursor to form the container, wherein the component is incorporatedin the container. 81-100. (canceled)
 101. A method, comprising:introducing a first polymer precursor into a mold, the mold having ashape configured to mold a container having a volume of at least 10 mL,the mold further comprising at least one mandrel for forming afunctional component of a liquid containment system; forming a containerwithin the mold; forming a component within the mold utilizing themandrel; and joining the functional component and the container withoutwelding. 102-107. (canceled)
 108. A method as in claim 107, wherein thewall portion of the container having a first thickness and the portionof the functional component having a second thickness which are joinedtogether have thicknesses within 20% of each other, relative to thelarger of the first and second thicknesses. 109-111. (canceled)
 112. Anarticle comprising: a collapsible bag comprising a flexible wall portionand a rigid portion comprising a base including a shaft configured tosupport a magnetic impeller, wherein the rigid portion is embedded withthe flexible wall portion. 113-124. (canceled)
 125. A method,comprising: introducing a first polymer precursor into a mold, the moldhaving a shape configured to mold a collapsible bag having a volume ofat least 10 mL and also configured to mold a base including a shaftconfigured to support a magnetic impeller; forming a collapsible bagwithin the mold; introducing a second polymer precursor into the mold;forming a component of the mixing system by solidifying the secondpolymer precursor; and joining the component of the mixing system andthe collapsible bag without welding.
 126. A method as in claim 125,wherein the first and second polymers are introduced into the moldsimultaneously. 127-133. (canceled)